School of Pharmacy, Anhui University of Chinese Medicine, Hefei, Anhui, 230012, China.
School of Pharmacy, Anhui University of Chinese Medicine, Hefei, Anhui, 230012, China; Anhui Province Key Laboratory of Chinese Medicinal Formula, Hefei, Anhui, 230012, China.
J Ethnopharmacol. 2021 Dec 5;281:114559. doi: 10.1016/j.jep.2021.114559. Epub 2021 Aug 28.
Naoluoxintong (NLXT) is a traditional Chinese Medicine (TCM) prescription that is clinically used in the treatment of ischemic stroke (IS). However, its therapeutic mechanism remains unclear.
To obtain the mechanism of NLXT by observing the protective effects of NLXT on the NogoA/RhoA/Rock pathway in a rat model of IS by regulating DNA methylation.
Rats were divided into five groups using a random number table: normal group, model group, NLXT group, blocker group I (NLXT + SGI-1027) and blocker group II (NLXT + Y27632). The right middle cerebral artery occlusion-reperfusion (MCAO/R) rat model was made, and the regional cerebral blood flow (rCBF) of each group was detected using laser Doppler. The methylation levels of CpG sites of neurite outgrowth inhibitor protein-A (Nogo-A), Nogo receptor (NgR), ras homolog gene family member A (RhoA) and rho-associated coiled-coil protein kinase 2 (ROCK2) genes in rat brain tissue were detected using the bisulfite method. Reverse transcription-polymerase chain reaction (RT-PCR) was used to detect NogoA, RhoA, NgR1, NgR2 and ROCK2 mRNA expression in rat brain tissue. NogoA, RhoA, NgR1, NgR2 and ROCK2 proteins were detected using immunoblotting in rat brain tissue.
After the modeling of middle cerebral artery occlusion (MCAO), neurological deficit test was made to ensure the success of the modeling. At each time point after surgery, the rCBF of the other groups decreased compared with the normal group (P < 0.01 or P < 0.05). Meanwhile, the rCBF increased in blocker group I as well as blocker group II after 3 days (P < 0.05). There were differences in the DNA methylation sites of NogoA, RhoA, NgR and ROCK2 genes between the model group and the NLXT group (P < 0.05). Compared with the normal group, NogoA, NgR1, NgR2, RhoA and ROCK2 gene expression in the model group increased observably (P < 0.01). In comparison with the model group, NogoA and NgR1 gene expression in the blocker group II was prominently observed on the 1st day. NogoA, NgR1, NgR2, RhoA and ROCK2 gene expression remarkably reduced (P < 0.01) on the 3rd and 7th days. Compared with the normal group, NogoA, RhoA, NgR1, NgR2 and ROCK2 protein expression in the model group increased observably (P < 0.01). In comparison with the model group, NogoA, RhoA, NgR1, NgR2 and ROCK2 protein expression in the other groups declined prominently (P < 0.01).
NLXT can reduce the DNA methylation level of NogoA pathway after IS, thus inhibit the expression of NogoA/RhoA/ROCK pathway from producing anti-cerebral ischemia pharmacological effect.
脑络通胶囊（NLXT）是一种中药方剂，临床上用于治疗缺血性中风（IS）。然而，其治疗机制尚不清楚。
通过观察 NLXT 通过调节 DNA 甲基化对 IS 大鼠模型中 NogoA/RhoA/Rock 通路的保护作用，获得 NLXT 的作用机制。
采用随机数字表法将大鼠分为五组：正常组、模型组、NLXT 组、阻滞剂 I 组（NLXT+SGI-1027）和阻滞剂 II 组（NLXT+Y27632）。制作右侧大脑中动脉闭塞再灌注（MCAO/R）大鼠模型，采用激光多普勒检测各组大鼠局部脑血流量（rCBF）。采用亚硫酸氢盐法检测大鼠脑组织中神经生长抑制蛋白-A（Nogo-A）、Nogo 受体（NgR）、ras 同源基因家族成员 A（RhoA）和 rho 相关卷曲螺旋蛋白激酶 2（ROCK2）基因的 CpG 位点甲基化水平。采用逆转录-聚合酶链反应（RT-PCR）检测大鼠脑组织中 NogoA、RhoA、NgR1、NgR2 和 ROCK2mRNA 的表达。采用免疫印迹法检测大鼠脑组织中 NogoA、RhoA、NgR1、NgR2 和 ROCK2 蛋白的表达。
MCAO 造模后进行神经功能缺损试验，以确保造模成功。术后各时间点，与正常组比较，其他各组 rCBF 均降低（P<0.01 或 P<0.05）。同时，阻滞剂 I 组和阻滞剂 II 组在术后 3 天 rCBF 增加（P<0.05）。模型组与 NLXT 组 NogoA、RhoA、NgR 和 ROCK2 基因的 DNA 甲基化位点存在差异（P<0.05）。与正常组比较，模型组 NogoA、NgR1、NgR2、RhoA 和 ROCK2 基因表达明显增加（P<0.01）。与模型组比较，阻滞剂 II 组第 1 天 NogoA 和 NgR1 基因表达明显降低，第 3、7 天 NogoA、NgR1、NgR2、RhoA 和 ROCK2 基因表达明显降低（P<0.01）。与正常组比较，模型组 NogoA、RhoA、NgR1、NgR2 和 ROCK2 蛋白表达明显增加（P<0.01）。与模型组比较，其他各组 NogoA、RhoA、NgR1、NgR2 和 ROCK2 蛋白表达明显降低（P<0.01）。
NLXT 可降低 IS 后 NogoA 通路的 DNA 甲基化水平，从而抑制 NogoA/RhoA/ROCK 通路的表达，产生抗脑缺血的药理作用。
